The transhydrogenase factor (TH) from R. rubrum has been purified by specific isolation from chromatophores, membrane, (NH4)2SO4 fractionation, Sephadex G200 chromatography and Amicon filtration. During the course of this purification it was found that the energy linked reaction (reaction I) I NADH plus NADP ion yields NAD ion plus NADPH catalyzed by the factor in the presence of resolved chromatophores (CT) and in an energy source (ATP, PPi or light) was lost (purification in the absence of dithiothreitol and EDTA) while that of the nonenergy linked reaction (reaction II) II NADH plus 3AcPyNAD ion yields NAD ion plus 3AcPyNADH was maintained (see below). It is reasoned that modification of SH groups on the factor prevents proper binding of TH plus CT and thus loss of catalysis of the energy linked reaction. The TH factors' ability to catalyze the nonenergy linked reaction is also lost upon purification; however this activity (in contrast to the energy dependent reaction) can be reconstituted by a small molecular weight component of a non-nucleotide nature. Our program for the grant period 04/01/77-03/31/78 involves: (a) Characterization of the activator for the nonenergy linked TH factor with respect to its chemical nature and the specificity of its interaction with TH factor. (b) Investigation of the specific modification of the TH factor which results in the loss of its ability to bind to CT membrane and consequent loss in the ability to catalyze the energy linked reaction. This probably involves modification of SH groups, or modification of an allosteric effector site. (c) The preparation of specific antibodies against the individual subunits of TH factor. One of the subunits is postulated as being involved in the binding to the CT membrane and antibodies against it could aid in the evaluation of that portion of CT which is involved with CT-TH interaction, i.e. the transducing portion of chromatophore membrane. (d) Arylazido beta alanine NAD ion, a photoaffinity label for the active site of pyridine nucleotide enzymes (Chen and Guillory, submitted to the JBC), will be used to further investigate the specificity of TH factor interaction with pyridine nucleotides. Of particular interest is the question as to which of the TH subunits is involved in pyridine nucleotide bind (Text Truncated - Exceeds Capacity)